[Show abstract][Hide abstract]ABSTRACT: To develop a singleplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella (S.) flexneri.
Eight pairs of primer for O-antigen synthesis and modification genes of S. flexneri were designed and used for developing an O-antigen modification gene-specific singleplex PCR assay to serotype 14 most common S. flexneri serotypes (1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Y, X, Xv and F6). Bacterial pathogens which causing diarrheal disease were used for specificity detection. 106 S. flexneri clinical isolates were serotyped by this method and compared with the slide agglutination method.
An O-antigen modification, gene-specific singleplex PCR was developed. When six singleplex PCR reactions were performed, 14 of the 15 recognized S. flexneri serotypes were identified, except for serotype Xv. The detection threshold ranged from 10 pg to 1 ng DNA in a 20 µl reaction system. A high concordance between the singleplex PCR assay and slide agglutination were observed when 106 S. flexneri strains of various serotypes were analyzed with an exception that 1 serotype Y strain showed that it was carrying the additional defective gtr II genes.
This method showed advantages over the traditional slide agglutination methods, and was promising when under application in the following situations as clinical diagnosis.

[Show abstract][Hide abstract]ABSTRACT: To establish a method combined morphology and molecular marker for identifying Haemaphysalis longicornis and Rhipicephalus microplus.
Ticks were collected from domestic animals and wild environment in epidemic area of Hubei and Henan provinces where cases of fever with thrombocytopenia syndrome were prevalent. We classified the ticks by morphology characteristics before 12S rDNA of ticks were amplified by PCR and subsequently sequenced. Phylogenetic tree was constructed by PAUP4.0.
The ticks belonged to Haemaphysalis longicornis and Rhipicephalus microplus through observation and analysed by the morphological characteristics of the ticks. 12S rDNA was cloned and sequenced while data confirmed the morphological identification of the results.
The method based on morphology that combined with molecular marker seemed a good method for the identificaton of ticks.

[Show abstract][Hide abstract]ABSTRACT: To study the integration site and arrangement of SfII and SfX prophages in Shigella flexneri serotype 2b strains.
A series of primers were designed based on potential integration site of SfII and SfX prophages in Shigella flexneri serotype 2b strains, and PCR were performed for 50 serotype 2b strains to amplify special genes located in host and prophages. PCR products were sequenced to identify integration sites and arrangement of SfII and SfX.
In all the serotype 2b strains, prophage SfII and SfX were adjacent to each other, and integrated into the thrW tRNA gene of the host, which were located between genes proA and yaiC of host. Prophage SfX was located immediately upstream of prophage SfII in all the detected 50 serotype 2b strains exception for strain 51251.
This was the first report on the integration site and arrangement of serotype-converting prophages SfII and SfX in Shigella flexneri 2b strains.

[Show abstract][Hide abstract]ABSTRACT: To develop a PFGE protocol for Streptococcus suis.
We developed and optimized a PFGE protocol for S. suis, in terms of plug preparation, choice of restriction endonucleases and optimized electrophoresis parameters. By analyzing the genome sequences of S. suis P1/7 with Mapdraw of DNAStar, we found three restriction enzymes, Swa I, Sma I and Apa I, were more suitable than others.
Analysis of 100 isolates of S. suis including 34 of 35 serotypes identified, 59, 53 and 43 patterns were obtained from Swa I, Sma I and Apa I restriction, respectively. The enzyme Swa I had the greatest power for discrimination ability.
By optimization of the protocol at various conditions, a rapid, reproducible, economic and practical PFGE method for S. suis was developed.

[Show abstract][Hide abstract]ABSTRACT: To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157 : H7 (EHEC O157 : H7) Shigela toxin 2B subunit (Stx2B) and vibrio cholera toxin B subunit (CTB) as well as to detect the immunogenicity and GM1-binding ability of fusion protein.
To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified, then to transform constructed plasmid into E. coli BL21 (DE3) induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDS-PAGE and Western-blot.
The amplified ctb-stx2b fragments appeared to be 750 bp and gene sequence was identical to designed sequence. The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about M(r) 20 x 10(3) and the expressed protein could react to CTB monoclone anti-body. The fusion protein CTB-Stx2B could bind GM1.
CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability. This study provided information on further EHEC O157 : H7 vaccine research.

[Show abstract][Hide abstract]ABSTRACT: To identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84.
Two-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005.
A total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005.
The newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.

[Show abstract][Hide abstract]ABSTRACT: To understand the epidemiological characteristics of enterohaemorrhagic Escherichia coli (EHEC) O157 and to determine the degree of its genetic relations.
Polymerase chain reaction (PCR) techniques and chromosomal DNA digested by restriction enzyme Xba I according to PulseNet directions by pulsed field gel electrophoresis (PFGE) method were applied to 300 E. coli O157 strains isolated from patients and animal sources from 1988 to 2005 from Henan, Jiangsu and Anhui provinces.
Very high prevalence of stx2 gene in EHEC O157:H7 strains isolated from some provinces of China was found and variation existed in some strains. We got 161 PFGE patterns from 300 strains. The stx2-producing strains could be clearly separated from stx2 variation-producing strains.
The variability of restriction enzyme-digestion patterns of O157 genomes suggested that the presence of some genomic diversity among the strains did exist.

[Show abstract][Hide abstract]ABSTRACT: To analyze the impact of depletion of the twin arginine translocation (TAT) system on virulence and physiology of Yersinia enterocolitica for a better understanding of its pathogenicity.
We constructed a DeltatatC::SpR mutant of Yersinia enterocolitica by P1 phage mediated transduction using Escherichia coli K-12 DeltatatC::SpR strain as a donor.
A P1-mediated genetic material transfer was found between the two species of enterobacteria, indicating a great potential of acquisition of antibiotic resistance in emergency of a new threatening pathogen by genetic material exchanges. Periplasmic trimethylamine N-oxidase reductase activity was detected in the wild type Y. enterocolitica strain and translocation of this enzyme was completely abolished by the DeltatatC::SpR mutation. In addition, the DeltatatC::SpR mutation showed a pleiotropic effect on the metabolism of Y. enterocolitica. However, the tat mutation did not seem to affect the mobility and virulence of Y. enterocolitica under the conditions used.
Unlike other pathogenic bacteria studied, the TAT system of Y. enterocolitica might play an important role in the pathogenic process, which is distinct from other pathogens, such as Pseudomonas aeruginosa and enterohemorrhagic E. coli O157:H7.

[Show abstract][Hide abstract]ABSTRACT: To understand the variation of Shiga toxin (stx) genes of Escherichia coli O157:H7 strains isolated in China.
Polymerase chain reaction (PCR) was used to identity the types of stx genes and the nucleotide sequences of the amplified stex variants genes were determined. Compare to the cytotoxicity of Stx,variants were tested by HeLa cell assay.
We found novel stx2 genes in 3 of 289 strains of Shiga toxin-producing E. coli O157:H7 isolated from 1999 to 2002 in China. The novel stx2 genes were inserted by a 1.3-kb insertion sequence (IS) and the nucleotide sequences of IS showed 100% homology with that of IS1203 variant (IS1203v). The IS1203v inserted in the stx2 genes of three E. coli O157:H7 strains at different sites and the direction of the open reading frames (ORFs) of IS1203v of each strain was different. In addition to the above mentioned findings, the nucleotide sequences of three stx2 genes were completely identical and the type of the three Stx2 was Stx2 prototype. Compare to the cytotoxicity of Stx2 prototype, the novel Stx2 was found to be obviously lower.
E. coli O157:H7 strains harboring stx2::IS1203v genes were isolated in China. Consequently, the results of HeLa cell assay showed that the insertion of IS1203v could lead to low cytotoxicity of Stx2.

[Show abstract][Hide abstract]ABSTRACT: To study the characteristics of epidemiology and molecular typing on Neisseria meningitidis serogroup C strains associated with outbreaks of Anhui province and sporadic cases in China, using pulsed field gel electrophoresis (PFGE).
212 Neisseria meningitidis serogroup C strains were isolated from invasive meningococcal cases, close contacts and healthy carriers, including 48 strains from Anhui province with 38 strains associated with serogroup C outbreaks. PFGE were performed by genomic DNA digestion with Nhe I restriction enzyme. The results of PFGE were analyzed by BioNumerics software (Version 4.0, Applied Maths BVBA, Belgium).
A total number of 212 Neisseria meningitidis serogroup C isolates were typed by 43 patterns, named AH1 to AH43. In China, AH1 pattern was the major PFGE pattern with 69.3% (n = 147) of all strains, distributed in 11 provinces. Three types of PFGE patterns (AH1 to AH3) were found in 48 strains from Anhui province, in which, 93.8% (n = 45) belonged to AH1. 97.4% (n = 37) of 38 strains associated with serogroup C outbreaks in Anhui province showed AH1 pattern. A total of 53 serogroup C strains were isolated from invasive meningococcal cases with 67.9% (36/53) of AH pattern. 71.9% (87/121) of serogroup C strains isolated from contacts of invasive meningococcal cases was AH1 pattern and 63.2% (24/38) of the strains from healthy carriers showed AH1 pattern.
By PFGE typing and analysis, AH1 pattern of Neisseria meningitidis serogroup C strains was proved to be the main clone which causing the outbreaks in Anhui province and might be responsible for the sporadic serogroup C meningococcal disease epidemics else where in the country.

[Show abstract][Hide abstract]ABSTRACT: To determine the genetic relationships between different Vibrio cholerae isolates in Shenzhen from 1993 to 2002.
Chromosomal DNA from 60 isolates was digested in seakem gold agrose with restriction enzyme Not I and plugs were then analyzed by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) patterns of V. cholerae isolates were clustered using BioNumerics software.
39 distinctive PFGE patterns were identified with each pattern having 20 to 30 bands. Most PFGE patterns were divided into cluster A or cluster B.
The closely related pandemic clone clusters of V. cholerae strains did exist in Shenzhen. PFGE of V. cholerae could be used for active surveillance and tracking for cholerae.

[Show abstract][Hide abstract]ABSTRACT: The Ministry of Public Health released the National Surveillance project on Shigellosis in August, 2005. This study was to reveal the antimicrobial resistance status of Shigella isolates through the National Shigellosis Surveillance System in 2005 in China, so as to provide evidence for the development of surveillance, prevention and cure of Shigellosis.
All the lab assistants received training from Chinese Center for Disease Control and Prevention. The project prescribed the uniform experimentation, quality control method, reagent, etc. Disc diffusion test(K-B) was carried out, following the CLSI methods. Data were analyzed by WHONET 5.4 software.
(1) 3 serotypes were identified and S. flexneri was common that accounted for 75.5% of all Shigella isolates followed by 24.4% of S. sonnei, but only 1 strain of S. dysenteriae was separated. (2) The resistant rates to tetracycline and ampicillin in Shigella spp were quite high, as over 90.0%. However, the resistant rate to Cefotaxime was the lowest, only 6.1%. The resistant rates were different between serotypes with the resistant rates of S. flexneri to ampicillin, ampicillin/clavulanate and ciprofloxacin were higher than those of S. sonnei (P < 0.001). (3) The multiple-antibiotic-resistance status in Shigella spp was quite serious and the resistant rate to five and more antimicrobials was 54.9%. The most common resistant patterns were seen on ampicillin, nalidixin, tetracycline and sulfamethoxazole. (4) There were some differences in subtypes and antimicrobial resistance among different provinces.
Cefotaxime seemed the best in curing Shigellosis at the clinic level. Programs regarding monitoring subtypes and antimicrobial resistance of Shigella should be in a continuous manner so as to understand the pathogens timely and to control the disease pertinently.

[Show abstract][Hide abstract]ABSTRACT: Emergence of severe acute respiratory syndrome (SARS) from the winter of 2002 to the spring of 2003 has caused a serious threat to public health.
To evaluate the safety and immunogenicity of the inactivated SARS coronavirus (SARS-CoV) vaccine, 36 subjects received two doses of 16 SARS-CoV units (SU) or 32 SU inactivated SARS-CoV vaccine, or placebo control.
On day 42, the seroconversion reached 100% for both vaccine groups. On day 56, 100% of participants in the group receiving 16 SU and 91.1% in the group receiving 32 SU had seroconverted. The geometric mean titre of neutralizing antibody peaked 2 weeks after the second vaccination, but decreased 4 weeks later.
The inactivated vaccine was safe and well tolerated and can elicit SARS-CoV-specific neutralizing antibodies.

[Show abstract][Hide abstract]ABSTRACT: To investigate the dynamic trend of specific antibody against severe acute respiratory syndrome (SARS)-CoV in serum collected at various periods among employees in Guangzhou Xinyuan animal market.
Volunteers from employees of the animal market were recruited and their serum specific antibody against SARS-CoV were determined by enzyme linked immunesorbent assay (ELISA) method.
Positive SARS-CoV specific IgG antibody was found 25.61% (n = 328), 13.03% (n = 238), 12.59% (n = 135), 5.04% (n = 139) and 9.43% (n = 53) among volunteers, which were sampled in May 2003, Dec. 2003, Jan. 2004, July 2004 and June 2005 respectively. No specific IgM antibody was found in all of those samples. Among 129 samples which were tested twice or more, 97 were all negative, 18 all positive, 13 changed from positive to negative but only one sample from negative to positive. When the volunteers were divided by the duration of their working experiences as short-term or long-term, those who had worked at animal market for less than or more then 6 months when being tested, the positive rate for long-term employees were relatively constant, however, all of the persons employed after January 2004, when the palm civets and raccoon dogs were culled from the market, were tested negative.
The prevalence of specific antibody against SARS-CoV in employees of the animal market were somehow related with the presence or absence of palm civet. No serum was tested positive for persons who were employed after palm civets and raccoon dogs were culled from market. This data indicated that the SARS-CoV might have been from the palm civets and raccoon dog, and the animal market seemed to serve as one of the sources of infection.

[Show abstract][Hide abstract]ABSTRACT: To investigate the epidemiological and molecular typing features of the pathogenic Yersinia enterocolitica strains isolated in China,using pulsed field gel electrophoresis(PFGE) and standardized PFGE method as well as typing database of Yersinia enterocolitica.
PFGE analysis was performed as Laboratory Directions for molecular subtyping of Salmonella by PFGE (PulseNet,USA) with some modifications and the results of PFGE were analyzed by BioNumerics soft (Version 4.0, Applied Maths BVBA, Belium).
114 O:3 Yersinia enterocolitica strains were typed by 25 patterns to have found that K6GN11C30012 (50 strains), K6GN11C30015(19 strains) and K6GN11C30016(10 strains) were the major patterns. K6GNllC30012 had 92.2% cluster similarity with K6GN11C30009-K6GN11C30023. This clone included 91.23% strains of 114 0:3 Yersinia enterocolitica strains. 51 0:9 Yersinia enterocolitica strains were typed by 14 patterns; K6GN11C90004 (22 strains) and K6GN11C90010 (13 strains)were the major patterns. K6GN11C90004 had 81.8% cluster similarity with K6GN11C90010 patterns. The major patterns of 0:3 and 0:9 serotypes were quite different.
O:3 Yersinia enterocolitica strains might originate from the same clone and had very few variation in different years and provinces but O:9 Yersinia enterocolitica strains from two different clones with some changes.

[Show abstract][Hide abstract]ABSTRACT: In mid-July 2005, five patients presented with septic shock to a hospital in Ziyang city in Sichuan, China, to identify the etiology of the unknown reason disease, an epidemiological, clinical, and laboratory study were conducted.
An enhanced surveillance program were established in Sichuan, the following activities were introduced: active case finding in Sichuan of (a) laboratory diagnosed Streptococcus suis infection and (b) clinically diagnosed probable cases with exposure history; supplemented by (c) monitoring reports on meningococcal meningitis. Streptococcus suis serotype 2 infection was confirmed by culture and biochemical reactions, followed by sequencing for specific genes for serotype and virulence factors.
From June 10 to August 21, 2005, 68 laboratory confirmed cases of human Streptococcus suis infections were reported. All were villagers who gave a history of direct exposure to deceased or sick pigs in their backyards where slaughtering was performed. Twenty six (38%) presented with toxic shock syndrome of which 15 (58%) died. Other presentations were septicaemia or meningitis. All isolates were tested positive for genes for tuf, species-specific 16S rRNA, cps2J, mrp, ef and sly. There were 136 clinically diagnosed probable cases with similar exposure history but incomplete laboratory investigations.
An outbreak of human Streptococcus suis serotype 2 infections occurred in villagers after direct exposure to deceased or sick pigs in Sichuan. Prohibition of slaughtering in backyards brought the outbreak to a halt. A virulent strain of the bacteria is speculated to be in circulation, and is responsible for the unusual presentation of toxic shock syndrome with high case fatality.

[Show abstract][Hide abstract]ABSTRACT: To study the distribution of Yersinia enterocolitica and its virulence factors in Nantong, Jiangsu.
Yersinia strains were isolated from livestock and poultry. Conventional PCR was used to detect the virulence factors of all strains and strain 0:8 was analyzed by pulsed-field gel electrophoresis(PFGE).
The combined isolation rate of Yersinia enterocolitica from livestock and poultry was 31.06% and the gene distribution characters were: 39.57% of them were ail-, ystA- , ystB-, yadA- , virF-; 60.43% were ail- , ystA- , ystB + , yadA- , virF- respectively. The two reference strains from America and Denmark showed similar electrophoresis patterns but were significantly different with O:8 strains isolated from China while the serotypes of Yersinia enterocolitica O:3 and O:9 which were the main epidemic strains in China, were not found in this area.
The pathogenic Yersinia enterocolitis O:3 and O:9 were not found in Nantong,Jiangsu province.